PROJECT SUMMARY This project is focused on developing a broad-spectrum antiviral drug for treating viral hemorrhagic fever diseases caused by Old World and New World arenaviruses. There are no FDA-approved therapeutics for treating infections caused by these viruses, and hemorrhagic fever arenaviruses are listed as NIAID Category A priority pathogens. Our small-molecule drug, LHF-535, is an investigational new drug (IND)-ready oral antiviral therapeutic against Lassa virus, an Old World arenavirus that causes Lassa fever. The drug targets the Lassa virus envelope glycoprotein and functions as a viral entry inhibitor. In this project, our goals are to assemble a data package to expand the labeled indication for LHF-535 to include infection with the New World hemorrhagic fever arenaviruses Junn virus and Machupo virus, address pre-IND recommendations by the FDA to more fully characterize the genotypic and phenotypic characteristics of arenaviruses that develop resistance to LHF-535, and formulate LHF-535 for intravenous (i.v.) administration. To achieve these goals, the following Specific Aims are proposed: 1) Define the antiviral activity of LHF-535 against New World hemorrhagic fever arenaviruses. Junn virus and Machupo virus are endemic in distinct regions of South America, where they cause sporadic outbreaks of severe disease with high morbidity and mortality. We will use well-established guinea pig infection models to define antiviral efficacy and to determine pharmacokinetic- pharmacodynamic relationships. 2) Determine the nature, emergence, and virulence of LHF-535-resistant viruses. We will evaluate the ability of LHF-535 to inhibit the infectivity of lentiviral pseudotypes expressing envelope glycoproteins of Junn virus that contain specifically engineered amino acid substitutions. Junn virus variants that are resistant to LHF-535 will also be identified by serial passage in LHF-535-treated cells and by isolating viruses from animals that fail LHF-535 treatment. To assist in monitoring the development of antiviral resistance during initial clinical efficacy studies, we will also generate and test an expanded library of genetic variants within the Lassa virus envelope glycoprotein. 3) Develop an i.v. formulation for LHF-535. Intravenous administration provides an option for drug delivery in situations where oral administration is not feasible. We will also explore the possibility that the rapid drug exposure provided by i.v. administration might enhance therapeutic efficacy, particularly in late stages of disease. Formulation development will include evaluation of antiviral efficacy (including fixed-dose and loading-dose strategies), toxicology, and safety pharmacology. Together, our studies will enhance the clinical value and efficacy of LHF-535 across multiple indications and support and inform Phase II clinical trials. LHF-535 is intended to both improve global health and serve as a medical countermeasure to the bioterrorism threat posed by hemorrhagic fever arenaviruses.